1. Field of the Invention
The invention relates generally to methods and apparatus for recovering specific cell populations from human bone marrow or cord blood. In particular, the invention includes a system and method for the high efficiency recovery (isolating and separating) of stem and progenitor cells, resident in bone marrow or cord blood, and the removal of excess red cells, neutrophils, platelets, and plasma, without the aid of xenobiotic additives typically employed to improve the efficiency of stem cell recovery. The system includes a sterile, functionally closed bag set and a microprocessor controlled electrical mechanical and optical device designed to operate in a centrifugal field that separates the cell populations based on their size and density and then transfers these stem and progenitor cells in a predetermined final volume to a stem cell bag. The invention also includes compositions of stem and progenitor cells prepared from human bone marrow or cord blood which would be for immediate use or for storage and later use.
2. Description of the Related Art
For purposes of this specification and the claims, the following definitions are used:
“Autologous use” means the implantation, transplantation, infusion, or transfer of human cells or tissue back into the individual from whom the cells or tissue were recovered.
“Crystalloid” means an isotonic salt and/or glucose solution used for electrolyte replacement or to increase intravascular volume, such as saline solution, Ringer's lactate solution, or 5 percent dextrose in water.
“Hematopoietic stem cell” is a pluripotent stem cell that gives rise to the many types of blood cells including red blood cells (RBCs), leukocytes, white blood cells (WBCs), and platelets.
“Leukocytes” or white blood cells are cells in the hematopoietic lineage which include principally monocytes, lymphocytes, and neutrophils and express the cell surface antigen CD45 (CD45+ cells).
“Lymphocytes” are cells of the lymphoid lineage as measured by the Sysmex XE-2100 which can include mature lymphocytes (T cells, B cells, NK cells) and developing lymphocytes such as lymphoblasts.
“Minimally manipulated bone marrow or cord blood” means the processing of anticoagulated (such as with heparin or citrate) bone marrow or cord blood for stem and progenitor cells that does not include the addition of chemical substances (except for water, crystalloids, or a sterilizing, preserving, or storage agents), and does not alter the relevant biological characteristics of the cells or tissues.
“Monocytes” are cells of the myeloid lineage as measured by the Sysmex XE-2100 which can include mature monocytes and developing monocytes such as monoblasts.
“Mononuclear cells” (MNC) are cells in the hematopoietic lineage which include monocytes and lymphocytes.
“Neutrophils” are cells of myeloid lineage as measured by the Sysmex XE-2100 which can include polymorphonuclear neutrophils and developing neutrophils such as the band form, metamyelocyte, myelocyte, and myeloblast.
“Platelets” are cells of the megakaryocyte line as measured by the Sysmex XE-2100 which can include mature platelets (thrombocytes).
“Progenitor cells” are a direct progeny of the stem cell that give rise to a distinct cell lineage by a series of cell divisions.
“Red cells” Cells of the erythroid line as measured by the Sysmex XE-2100 which include mature RBC but not nucleated red cells.
“Stem cells” are pluripotent cells that have three general properties: (1) they are capable of dividing and renewing themselves for long periods; (2) they are unspecialized; and (3) they can give rise to different specialized cell types. A cell surface antigen marker for stem cells is the CD34 antigen as is the elevated concentration of the intracellular enzyme aldehyde dehyrogenase (ALDH).
“Stoke's Law” is a mathematical formula (Vg=d2(P1−P2)/18μ×G) that describes the sedimentation velocity of a particle in a viscous liquid during gravitational acceleration, wherein:                Vg=sedimentation velocity,        d=particle diameter,        P1=particle density,        P2=liquid density,        G=gravitational acceleration, and        μ=viscosity of liquid.        
“Sysmex XE-2100” is an automated hematology cell analyzer manufactured by Sysmex Corp, Kobe, Japan that differentiates and quantitates hematopoietic cells by flow cytometry, emitting a semiconductor laser beam, and detecting three optical signals: forward scatter, side scatter, and side fluorescence.
“Total nucleated cells” (TNC) are WBCs and nucleated RBCs.
“Xenobiotic additive” and “xenobiotic agent” mean a chemical substance which is not a natural component of the human body that is intentionally added to the collected bone marrow or cord blood for the purpose of achieving a change in the cell separation process performance characteristics that would not occur if it was not added.
Examples of xenobiotic additives are:
1. sedimentation agents (e.g., hydroxy ethyl starch, dextran, or gelatins);
2. density gradient media (e.g., Ficoll®, Percoll®); and
3. affinity molecules for cell surface macromolecules cells (e.g., monoclonal antibodies, monoclonal antibodies bound to paramagnetic beads).
To date, despite the enormous clinical potential of stem cells, no stem cell therapies have received FDA clearance for human use. A major barrier to demonstrating clinical efficacy and safety and obtaining regulatory clearance of a stem cell therapy is that the existing devices and methods used to separate the stem cell populations from bone marrow or cord blood have one or more of the following significant limitations: they are open systems risking microbial contamination; they are time and labor intensive; they require the addition of undesirable and expensive xenobiotic agents; they recover stem cells at a very low efficiency, averaging 40 to 75%; and they are not compatible with the volume of cord blood or bone marrow utilized for tissue regeneration.
Before broad scale use of stem cells to treat serious and frequent human diseases, such as myocardial infarction, ischemia, diabetes and dermal wounds can occur, three critical hurdles must be overcome:
1. Clinical trials must demonstrate efficacy and safety of the stem cell therapy;
2. Regulatory clearance by government health agencies, in particular, the Food and Drug Administration (FDA) Office of Cell, Tissue and Gene Therapy (OCTGT) in the United States, must be obtained; and
3. A practical method for the rapid and simple recovery of viable stem and progenitor cells from minimally manipulated bone marrow or cord blood that does not rely on the use of xenobiotic additives to remove excess plasma, red cells, and granulocytes must be developed.
In addition to hematopoietic stem cells, it is now known that bone marrow and cord blood contain other types of stem and progenitor cells of potential therapeutic value for tissue regeneration and to enhance wound healing. Mesenchymal stem cells previously designated as bone marrow stromal cells can give rise to bone, cartilage, fat, muscle, and connective tissue and are ALDH Br+. Endothelial progenitor cells, resident in the bone marrow, are primitive cells descended from hematopoietic stem cells, can enter the bloodstream and go to areas of blood vessel injury to help repair the damage, can give rise to new blood vessels, and are CD34+.
Stem cells are identified by various cell surface markers, including CD34+ and aldehyde dehydrogenase bright cells (ALDH Br+). CD34+ cells are stem cells that express an antigen designated as CD34 which can be detected by using a chromophore conjugated antibody with specificity to that particular antigen. Other researchers identify adult stem cells based upon their expressing high levels of the enzyme aldehyde dehydrogenase (ALDH). One company, Aldagen (Durham, N.C.) developed a technology which utilizes a substrate that detects cells expressing high levels of ALDH by generating an intense green fluorescence. These so-called ALDH bright cells (ALDH Br+) can be detected using flow cytometry. ALDH Br+ cells contain different progenitors that can give rise to a variety of important cell lines, including neurological and endothelial cells.
Viability of stem cells can be demonstrated by several methods including exclusion of a vital dye such as trypan blue or 7-amino-actinomycin D (7-AAD). Viability of stem cells is routinely assessed by measuring the viability of white blood cells (CD45+ cells) as a surrogate for the stem cells in the bone marrow or cord blood using commercially available kits.
Large volumes of bone marrow or cord blood are unwieldy, especially because of the presence of a large number of red cells (there are approximately 25,000 red cells for each CD34+ cell in bone marrow and approximately 180,000 red cells for each CD34+ cell in cord blood). These excess red cells make it difficult to use the stem and progenitor cells because their presence radically dilutes the concentration of stem and progenitor cells at a wound site requiring tissue regeneration and they interfere with other processing steps for stem cells, including cultivating for ex-vivo cell expansion, gene therapy, or affinity purification of cell populations. Further, the presence of such a large number of red cells raises other safety issues, such as ABO incompatibility in allogeneic transplantation. In some cases, it may also be desirable to minimize neutrophils from the volume reduced stem cell concentrate due to the potential association of these cells with inflammatory reactions and the presence of biologically active enzymes and cytokines in their cytoplasm that could affect the viability or plasticity or otherwise change the functionality of stem cells.
The rate of movement and final location of a cell after a fixed period of centrifugation is a function of its size and density with its movement being defined according to Stoke's Law. Stem cells are lower in density than RBCs and higher in density than platelets, having a density closer to that of WBCs. Thus, the conventional method of recovering stem cells by density and size typically involves stratifying the cell population by centrifugation and then, after removal from the centrifuge, attempting to capture the stem cells from the remainder of the cord blood or bone marrow.
One manual method of isolating WBCs and stem cells from bone marrow or cord blood is to insert whole bone marrow or cord blood into centrifuge tubes and spin them at 1500-2000×g for 10-15 min at room temperature. This separates the bone marrow or cord blood into an upper plasma layer, a lower red blood cell (RBC) layer, and a thin layer of WBCs and stem and progenitor cells at the interface between the RBC and plasma. After the stratification is accomplished, a disposable, plastic transfer pipette can be used to aspirate off the plasma (upper layer) down to about 1 mm from the RBCs. When removing the plasma, great care must be taken to not disturb the WBC/stem and progenitor cell layer which must be removed by the same pipetting technique while attempting to guard against loss of stem and progenitor cells into the RBCs or excessive contamination of the stem cells with RBCs. The disadvantages of this manual approach in test tubes are that it is an open system which risks microbial contamination of the bone marrow unit; it is labor and time intensive; the amount of red cell contamination of the WBC/stem and progenitor cell layer is highly variable; and the loss of stem cells is significant and variable. Microbial contamination, a major concern with this method, is important to avoid as it can negatively impact the ability to culture the stem cells or risk the transmission of infection to the body of the recipient of the stem cells.
Another method of recovering WBC/stem and progenitor cell populations from cord blood or bone marrow utlizes cell processing equipment such as the Compomat G4 device (Fresenius Kabi AG, Friedberg, Germany) and the COBE 2991 Blood Cell Processor (COBE Laboratories, Lakewood, Colo.). An example of this type of process is presented in Tsubaki, et al. “Concentration of Progenitor Cells Collected from Bone Marrow Fluid Using a Continuous Flow Cell Separator System”, Apheresis and Dialysis 5 (1), 46-48, 2001. The amount of bone marrow or cord blood harvested for stem cells depends on the proposed clinical use. For cell based therapies in tissue regeneration or repair, typically 50-150 mL of bone marrow or cord blood is collected. These blood bank instruments require large volumes of bone marrow or cord blood (over 200 mL) to function properly and are entirely unsuited for the low volumes typically collected for tissue regeneration medical applications (less than 200 mL). In addition, the final volume of the cellular product (50-100 mL) of these mechanized processes is substantially larger than the preferred, final volume of 3-20 mL that provides the concentration of stem and progenitor cells appropriate for clinical use. These instruments also require pumps to move the blood or bone marrow from one container to another and these pumps can potentially cause mechanical damage to the cells.
Another method for volume reduction and purification of bone marrow or cord blood utilizes a gradient media such as Ficoll® or Percoll® to combine with the bone marrow or cord blood and, during centrifugation, interpose itself between the cell populations to reduce the mixing of cell populations during attempts to recover the stem cells. Ficoll® is a neutral, highly branched, high-mass, hydrophilic polysaccharide. Ficoll® (e.g. density of 1.077) can be used to separate blood or bone marrow into its components (erythrocytes, leukocytes, etc.). Ficoll® is normally placed at the bottom of a tube, and bone marrow or cord blood diluted with saline is then slowly layered above the Ficoll®. After being centrifuged, the heavier RBCs (density 1.09-1.10) displace the Ficoll® at the bottom of the tube and the following layers will be visible in the column, from top to bottom: (1) plasma and platelets; (2) mononuclear cells (MNC) (density 1.06-1.07); (3) Ficoll®; and (4) erythrocytes and neutrophils (density 1.08-1.09) present in pellet form at the bottom. This separation allows a harvest of MNCs with less chance of co-mingling the MNCs with the RBCs. Some red blood cell trapping (presence of erythrocytes and neutrophils) still occur in the MNC layer.
A significant disadvantage of processing bone marrow or cord blood with Ficoll® is that it is an open system at the step of adding the Ficoll® to the bone marrow or cord blood, meaning that microbes can enter directly into the bone marrow or cord blood and thereby increase the risk of microbial contamination. To reduce this risk, it is necessary to perform this step using a biological safety cabinet which may not always be available and is an expensive piece of laboratory equipment that must be continually maintained and monitored for acceptable performance. Further, Ficoll® is a xenobiotic and must be removed by washing before the cells can be safely administered to the human body. This washing step requires time and effort and further increases the potential for microbial contamination and loss of cells. The recovery of stem cells is low and highly variable, ranging from 20 to 70%. Percoll®, a colloidal silica coated with polyvinylpyrrolidone, is another density gradient media similar to Ficoll® with its attendant disadvantages.
A technique for isolation of bone marrow white blood cells using hydroxyethyl starch (HES) sedimentation agent has been published by A. Montuoro et al., “A technique for isolation of bone marrow cells using hydroxyethyl starch (HES) sedimentation agent,” Haematologica 76 Suppl 1:7-9, 1991. HES is used clinically as a plasma expander and is characterized by its molecular weight and its degree of substitution. The addition of HES typically in a final concentration of 0.5 to 2% weight of HES to volume of bone marrow or cord blood causes the agglutination of red cells and thereby changes their sedimentation velocity based upon the principals of Stoke's Law. However, HES is a xenobiotic and has been associated with adverse events in some patients when administered intravenously. Further, the use of HES adds cost and additional complexity to cell processing. Dextran polymer preparations and gelatin solutions have also been used for the same purpose and have the same disadvantages as HES.
Finally, it is also known that stem and progenitor cells can be isolated from bone marrow or cord blood by the use of immunological methods, including flow cytometry cell sorting (Beckton Dickinson or Coulter) and by antibody coated paramagnetic particle cell sorting such as the Miltenyi CliniMacs or Baxter Isolex systems. Antibodies with specificity to stem cell antigens, such as CD34, are employed as part of these separation technologies. These methods have the disadvantage of requiring expensive xenobiotic agents, equipment and disposable processing sets and are time consuming to perform. While the final product is a more pure population of stem cells with little red cell or leukocyte contamination, this level of purity represents a significant incurred expense and may not be required to achieve the intended cell therapy effect. Further, the addition of the antibodies and beads to the bone marrow or cord blood means the product was not “minimally manipulated” during the process as these reagents may cause unintended adverse biological consequences such as irreversible stem cell differentiation into lineage committed pathways.
A study by Minguell, et al. “Preparative separation of nucleated cells from human bone marrow”, Experentia 35:548-549, 1978, compared volume reduction, cell recovery, and cell viability as a result of processing with dextran, dextran plus Ficoll®, Ficoll® and a buffy coat method. The paper demonstrates the lack of an ideal preparative process for bone marrow in that some of the processes caused loss of cell viability, and all had relatively low cell recovery of lymphoid cells (monocytes and lymphocytes or MNC) of 68% or less. To achieve the observed erythrocyte (RBC)/nucleated cell ratio of 2.7:1, the centrifugation method without sedimentation aid designated as the buffy coat method had a range of lymphoid cell recovery of these cells initially present that was low and highly variable, ranging from 36 to 68%.